The
Tet38 membrane protein has distinct functions, including drug efflux and host cell attachment and internalization mediated by interaction with host cell CD36. Using structural modeling and ...site-directed mutagenesis, we identified key amino acids involved in different functions. Tet38, a member of the major facilitator superfamily, is predicted to have 14 transmembrane segments (TMS), 6 cytoplasmic loops, and 7 external loops. Cysteine substitutions of arginine 106 situated at the junction of TMS 4 and external loop L2, and glycine 151 of motif C on TMS 5, resulted in complete or near-complete (8- to 16-fold) reductions in Tet38-mediated resistance to tetracycline, with minimal to no effect on A549 host cell internalization. In contrast, a three-amino-acid deletion, F
P
G
, in external loop L7 situated between TMS 13 and 14 led to a decrease of 4-fold in
internalization by A549 cells and a partial effect on tetracycline resistance (4-fold reduction). A three-amino-acid deletion, D
D
L
, in external loop L1 situated between TMS-1 and TMS-2, had a similar partial effect on tetracycline resistance but did not affect cell internalization. Using an Ni column retention assay, we showed further that the L7, but not the L1, deletion impaired binding to CD36. Thus, the L7 domain of Tet38 is key for interaction with CD36 and host cell internalization, and amino acids R
and G
(TMSs 4 and 5) are particularly important for tetracycline resistance without affecting internalization.
Five bacterial targets have been exploited in the development of antimicrobial drugs: cell wall synthesis, protein synthesis, ribonucleic acid synthesis, deoxyribonucleic acid (DNA) synthesis, and ...intermediary metabolism. Because resistance to drugs that interact with these targets is widespread, new antimicrobials and an understanding of their mechanisms of action are vital. The fluoroquinolones are the only direct inhibitors of DNA synthesis; by binding to the enzyme-DNA complex, they stabilize DNA strand breaks created by DNA gyrase and topoisomerase IV. Ternary complexes of drug, enzyme, and DNA block progress of the replication fork. Cytotoxicity of fluoroquinolones is likely a 2-step process involving (1) conversion of the topoisomerase-quinolone-DNA complex to an irreversible form and (2) generation of a double-strand break by denaturation of the topoisomerase. The molecular factors necessary for the transition from step 1 to step 2 remain unclear, but downstream pathways for cell death may overlap with those used by other bactericidal antimicrobials. Studies of fluoroquinolone-resistant mutants and purified topoisomerases indicate that many quinolones have differing activities against the two targets. Drugs with similar activities against both targets may prove less likely to select de novo resistance.
Chiral nano- or metamaterials and surfaces enable striking photonic properties, such as negative refractive index and superchiral light, driving promising applications in novel optical components, ...nanorobotics, and enhanced chiral molecular interactions with light. In characterizing chirality, although nonlinear chiroptical techniques are typically much more sensitive than their linear optical counterparts, separating true chirality from anisotropy is a major challenge. Here, we report the first observation of optical activity in second-harmonic hyper-Rayleigh scattering (HRS). We demonstrate the effect in a 3D isotropic suspension of Ag nanohelices in water. The effect is 5 orders of magnitude stronger than linear optical activity and is well pronounced above the multiphoton luminescence background. Because of its sensitivity, isotropic environment, and straightforward experimental geometry, HRS optical activity constitutes a fundamental experimental breakthrough in chiral photonics for media including nanomaterials, metamaterials, and chemical molecules.
The fluoroquinolones interact with 2 bacterial targets, the related enzymes DNA gyrase and topoisomerase IV, both of which are involved in DNA replication. Quinolones form complexes of these enzymes ...with DNA, complexes that block movement of the DNA-replication fork and thereby inhibit DNA replication. Many older quinolones differ in their relative activities against gyrase and topoisomerase IV in a bacterial cell, having greater potency against gyrase than against topoisomerase IV in many gram-negative bacteria and greater potency against topoisomerase IV than against gyrase in many gram-positive bacteria. Several newer quinolones appear to have more closely balanced activity against these enzymes. Resistance to fluoroquinolones occurs as a result of mutational amino acid substitutions in the subunits of the more sensitive (or primary-target) enzyme within the cell. If, however, both enzymes are similarly susceptible to a fluoroquinolone, then the level of resistance caused by a primary-target mutation may be low and may be limited by the sensitivity of the secondary target. Fluoroquinolones also differ in the extent to which common bacterial multidrug efflux pumps affect their activity, with some compounds being unaffected by resistance mechanisms because of overexpression of such pumps. Newer fluoroquinolone interaction with dual targets and avoidance of efflux-resistance mechanisms may each contribute to the lower frequencies of selection of resistant mutants in the laboratory.
Broad use of fluoroquinolones has been followed by emergence of resistance, which has been due mainly to chromosomal mutations in genes encoding the subunits of the drugs' target enzymes, DNA gyrase ...and topoisomerase IV, and in genes that affect the expression of diffusion channels in the outer membrane and multidrug-resistance efflux systems. Resistance emerged first in species in which single mutations were sufficient to cause clinically important levels of resistance (e.g., Staphylococcus aureus and Pseudomonas aeruginosa). Subsequently, however, resistance has emerged in bacteria such as Campylobacter jejuni, Escherichia coli, and Neisseria gonorrhoeae, in which multiple mutations are required to generate clinically important resistance. In these circumstances, the additional epidemiologic factors of drug use in animals and human-to-human spread appear to have contributed. Resistance in Streptococcus pneumoniae, which is currently low, will require close monitoring as fluoroquinolones are used more extensively for treating respiratory tract infections.
Background. Community-associated methicillin-resistant S. aureus (CA-MRSA) is the most common organism isolated from purulent skin infections. Antibiotics are usually not beneficial for skin abscess, ...and national guidelines do not recommend CA-MRSA coverage for cellulitis, except purulent cellulitis, which is uncommon. Despite this, antibiotics targeting CA-MRSA are prescribed commonly and increasingly for skin infections, perhaps due, in part, to lack of experimental evidence among cellulitis patients. We test the hypothesis that antibiotics targeting CA-MRSA are beneficial in the treatment of cellulitis. Methods. We performed a randomized, multicenter, double-blind, placebo-controlled trial from 2007 to 2011. We enrolled patients with cellulitis, no abscesses, symptoms for <1 week, and no diabetes, immunosuppression, peripheral vascular disease, or hospitalization (clinicaltrials.gov NCT00676130). All participants received cephalexin. Additionally, each was randomized to trimethoprim-sulfamethoxazole or placebo. We provided 14 days of antibiotics and instructed participants to continue therapy for ≥1 week, then stop 3 days after they felt the infection to be cured. Our main outcome measure was the risk difference for treatment success, determined in person at 2 weeks, with telephone and medical record confirmation at 1 month. Results. We enrolled 153 participants, and 146 had outcome data for intent-to-treat analysis. Median age was 29, range 3–74. Of intervention participants, 62/73 (85%) were cured versus 60/73 controls (82%), a risk difference of 2.7% (95% confidence interval, −9.3% to 15%; P = .66). No covariates predicted treatment response, including nasal MRSA colonization and purulence at enrollment. Conclusions. Among patients diagnosed with cellulitis without abscess, the addition of trimethoprim-sulfamethoxazole to cephalexin did not improve outcomes overall or by subgroup. Clinical Trials Registration. NCT00676130.
Fosfomycin inhibits MurA following uptake by the GlpT transporter of glycerol-3-phosphate in
In
, plasmid overexpression of the Tet38 efflux pump and a
mutant resulted in increased MICs and decreased ...accumulation of fosfomycin, with MICs affected by glycerol-3-phosphate. In contrast, a
mutant had a lower MIC and increased accumulation of fosfomycin, suggesting that Tet38 acts as an efflux transporter of fosfomycin.
Using an affinity column retention assay, we showed that the purified Tet38 membrane transporter of
bound specifically to host cell CD36 and to the complex CD36-Toll-like receptor 2 (TLR-2), but not ...to TLR-2 alone or TLR-2 and
lipoteichoic acid (LTA). We tested the effect of LTA on the internalization of
mutant QT7 versus RN6390 by A549 epithelial cells. Addition of anti-LTA antibody to the bacteria prior to adding to A549 cells reduced internalization of QT7 2-fold compared to that with nonspecific antibody treatment. QT7 internalized 4- to 6-fold less than RN6390 with or without anti-LTA antibody. These data suggested that Tet38 and LTA were independently involved in the invasion process. The wall teichoic acid (WTA) inhibitor tunicamycin had an 8-fold decrease in activity with overexpression of
and a 2-fold increase in activity in QT7 (
). Reserpine (an inhibitor of efflux pumps) reduced the effect of
overexpression on tunicamycin resistance 4-fold. In addition,
affected growth in the presence of LTA inhibitor Congo red, with overexpression increasing growth and deletion of
reducing growth. In conclusion, Tet38 contributes to
invasion of A549 via direct binding to CD36 of the complex CD36-TLR-2, and LTA independently bound to TLR-2. The reduction of tunicamycin resistance in the presence of reserpine and the survival ability of the
overexpressor in the presence of Congo red suggest that Tet38 can also protect the synthesis of LTA and WTA in
against their inhibitors, possibly functioning as an efflux pump.
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